Since its appearance in 2021, Alphafold2, an accurate protein structure prediction method, has quickly been adopted and substantially impacted biology research, particularly in the field of structural biology. In this review, we discuss recent advancements in the development of computational structural modeling tools related to Alphafold2. Additionally, we discuss deep learning-based structure modeling methods for cryo-electron microscopy.

The overall quality of the experimentally determined structures contained in the PDB is exceptionally high, mainly due to the continuous improvement of model building and structural validation programs. Improving reproducibility on a large scale requires expanding the concept of validation in structural biology and all other disciplines to include a broader framework that encompasses the entire project. A successful approach to science requires diligent attention to detail and a focus on the future. An earnest commitment to data availability and reuse is essential for scientific progress, be that by human minds or artificial intelligence.

The fragment molecular orbital（FMO）method is a theoretical method that enables quantum chemical calculations of whole bio-macromolecules, such as proteins and nucleic acids, yielding the energies and electron densities of whole molecules as well as interaction energies between fragments. The FMO calculations of protein complexes that have been structurally analyzed experimentally allow for quantitative analysis and physicochemical interpretation of intra- and intermolecular interactions within the complexes. Here we describe the quantitative analysis of structures and interactions using the FMO method, i.e., the “structure and interaction basis” obtained through the complementary use of experimental structure and the FMO calculations.

The resolution of the optical microscope was limited by diffraction. Recent advances in the super-resolution microscope have broken this barrier. In this review, its limitations and future perspectives are discussed.

As for the transmission muon microscopes（TμM）which are being developed at J-PARC, their principle, purpose, design and applications are reported. The TμM is an analog of a transmission electron microscope（TEM）, it employes muon beams generated by an accelerator, instead of the electron beams on TEM. The excellent material penetrating ability of accelerated muon beams allowed it to observe quite thick objects in high-resolutions. In particular, the TμM visualizes electromagnetic field distribution inside the thick bulk samples by using the Lorentz methods and the phase-contrast method. The TμM will be applied to a wide range of fields, not only academia but also industry.

Small-angle X-ray scattering（SAXS）for solution samples of biological macromolecules, recently called BioSAXS, has been used to estimate the conformational state of flexible proteins and unstable protein complexes in solution. With the advent of a new technique called SEC-SAXS, it has become easier to obtain structural information derived only from the target molecule, and it is now possible to estimate the structural state in solution with higher accuracy than 10 years ago. In this article, I will introduce not only some characteristic examples of BioSAXS analysis, but also the recently developing technique of time-resolved measurement.

The paradigm shift in drug discovery that began in the early 1990s was the development of Structure Based Drug Design（SBDD）as a means of rational drug design for disease targets. The greatest advantage of X-ray analysis is that once the crystal structure is obtained, structural information can be fed back very quickly under almost the same conditions for soaking and co-crystallization, contributing to lead optimization in the early stages of drug discovery. In addition, it is unique in that it covers target molecules from small molecular weights（20-30 kDa）to large molecular weights（100 kDa～）. On the other hand, it has many weak points such as dynamic structure close to the physiological state, identification of hydrogen atoms, and acquisition of charge information, which, when combined with multiple methods in a complementary manner, will contribute to a significant reduction in the lead optimization period in the future.

In modern life science research, structural information on proteins is indispensable for understanding the mechanisms of their biological functions. Many protein structures have been determined by X-ray crystallography, which has been developed in conjunction with molecular biology experiments and synchrotron radiation development. Since the Nobel Prize in Chemistry in 2017, the rapid progress of cryo-EM hardware and analysis software has led to the use of cryo-EM single-particle analysis for structural analysis. In this article, we compare the features of protein crystallography and cryo-EM single-particle analysis and discuss the best method for protein crystallography.